Intracellular Calcium and Myosin Isoform Transitions

Intracellular calcium levels can have profound effects on muscle biology via alterations in gene expression. In particular, intracellular calcium levels increase during muscle activation and are thought to underlie fast-to-slow shifts in muscle gene expression. In the present work, we determined that increased intracellular calcium has a significant effect on the activity of the adult fast myosin heavy chain (MyHC) promoters in the order of MyHC IIa ≫ IId/x > IIb. We have identified the pathways by which the calcium signal mediates increased activation of the MyHC IIa promoter. Inhibition of calcineurin or calcium-calmodulin kinase greatly attenuates ionophore-induced activation of the MyHC IIa promoter, whereas protein kinase C inhibitors have no effect. Inhibition and overexpression studies with members of the mitogen-activated protein kinase family reveal roles for MEK1/MEK2 and MEKK1, but not p38 or phosphatidylinositol 3-kinase. Downstream mediators of these effects are the activities of the MEF-2 and NFAT transcription factors, whose binding sites in the MyHC IIa promoter are required for calcium-induced activation of the MyHC IIa promoter. Intracellular calcium levels can have profound effects on muscle biology via alterations in gene expression. In particular, intracellular calcium levels increase during muscle activation and are thought to underlie fast-to-slow shifts in muscle gene expression. In the present work, we determined that increased intracellular calcium has a significant effect on the activity of the adult fast myosin heavy chain (MyHC) promoters in the order of MyHC IIa ≫ IId/x > IIb. We have identified the pathways by which the calcium signal mediates increased activation of the MyHC IIa promoter. Inhibition of calcineurin or calcium-calmodulin kinase greatly attenuates ionophore-induced activation of the MyHC IIa promoter, whereas protein kinase C inhibitors have no effect. Inhibition and overexpression studies with members of the mitogen-activated protein kinase family reveal roles for MEK1/MEK2 and MEKK1, but not p38 or phosphatidylinositol 3-kinase. Downstream mediators of these effects are the activities of the MEF-2 and NFAT transcription factors, whose binding sites in the MyHC IIa promoter are required for calcium-induced activation of the MyHC IIa promoter. Mammalian skeletal muscle consists of a mosaic of different fiber types. Four distinct fiber types, one slow (type I) and three fast (IIa, IId/x, and IIb), are found in adult rodent skeletal muscle, and these differ with respect to their fatigability and their strength and speed of contraction. The functional, biochemical, and morphological differences between muscle fiber types arise as a result of different programs of fiber-specific gene expression that result in specific contractile, metabolic, cytoskeletal, and regulatory proteomes associated with each fiber type. Skeletal muscle fibers demonstrate remarkable plasticity, and shifts in fiber type can occur in response to a number of physiological and pathological conditions, including muscle adaptation, aging, and muscle disease (1Pette D. Staron R.S. Int. Rev. Cytol. 1997; 170: 143-223Google Scholar). An increase in muscle activity such as endurance exercise training induces muscle fiber adaptations through qualitative and quantitative changes in fiber-specific contractile and metabolic gene expression that result in slower contracting, more oxidative muscle fibers (2Pette D. Med. Sci. Sports Exerc. 1984; 16: 517-528Google Scholar). The stimuli that induce these shifts in fiber-specific gene expression are currently not well defined. Likely candidates include many of the consequences of increased or prolonged muscle activation, including changes in the amount or pattern of muscle mechanical and electrical activity; changes in intracellular metabolites such as glycogen/glucose levels, hydrogen ions, or reactive oxygen species; and increased secretion of autocrine/paracrine factors. The amount and pattern of electrical activity induced in muscle fibers by the motoneuron have been postulated to play a prominent role in defining muscle gene expression and fiber type (3Buonanno A. Fields R.D. Curr. Opin. Neurobiol. 1999; 9: 110-120Google Scholar). One of the most critical alterations in response to electrical activation of cells is an increase in intracellular calcium levels. Depolarization-induced increases in intracellular calcium and its effects on gene expression have been studied in a number of electrically active cell types, including cardiac myocytes, neurons, and pancreatic islet cells (4Miranti C.K. Ginty D.D. Huang G. Chatila T. Greenberg M.E. Mol. Cell. Biol. 1995; 15: 3672-3684Google Scholar, 5Johnson C.M. Hill C.S. Chawla S. Triesman R. Bading H. J. Neurosci. 1997; 17: 6189-6202Google Scholar, 6McDonough P.M. Hanford D.S. Sprenkle A.B. Mellon N.R. Glembotski C.C. J. Biol. Chem. 1997; 272: 24046-24053Google Scholar, 7Bernal-Mizrachi E. Wice B. Inoue H. Permutt M.A. J. Biol. Chem. 2000; 275: 25681-25689Google Scholar, 8West A.E. Chen W.G. Dalva M.B. Dolmetsch R.E. Kornhauser J.M. Shaywitz A.J. Takasu M.A. Tao X. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11024-11031Google Scholar). These studies have also identified several signaling molecules involved in transducing the calcium signal, including serum response factor (SRF), 1The abbreviations used are: SRF, serum response factor; MyHC, myosin heavy chain; MEF-2, myocyte-specific enhancer factor 2; NFAT, nuclear factor/activator of T cells; CMV, cytomegalovirus; CaM kinase, calcium-calmodulin kinase; HDAC, histone deacetylase; MAP, mitogen activated protein; MEKK1, mitogen-extracellular kinase kinase-1. cyclic AMP-response element-binding protein, protein kinase C, and various members of the MAP kinase signaling family. Not surprisingly, several skeletal muscle genes are sensitive to electrical activity and/or intracellular calcium levels. Electrical stimulation of myotubes in culture up-regulates glycogen phosphorylase (9Vali S. Carlsen R. Pessah I. Gorin F. J. Cell. Physiol. 2000; 185: 184-199Google Scholar) but represses expression of the acetylcholine receptor (10Altiok N. Changeux J.-P. FEBS Lett. 2001; 487: 333-338Google Scholar) and fast sarcoplasmic ATPase genes (11Thelen M.H.M. Simonides W.S. Hardeveld C. Biochem. J. 1997; 321: 845-848Google Scholar). Increases in intracellular calcium up-regulate several skeletal muscle genes, including the myogenic regulatory factor Myf-5, the inward rectifying potassium channel, and the metabolic enzymes hexokinase II and cytochrome c(12Friday B.B. Pavlath G.K. J. Cell Sci. 2001; 114: 303-310Google Scholar, 13Shin K.S. Park J.-Y. Kwon H. Chung C.H. Kang M.-S. J. Biol. Chem. 1997; 272: 21227-21232Google Scholar, 14Freyssenet D. Di Carlo M. Hood D.A. J. Biol. Chem. 1999; 274: 9305-9311Google Scholar, 15Halseth A.E. O'Doherty R.M. Printz R.L. Bracy D.P. Granner D.K. Wasserman D.H. J. Appl. Physiol. 2000; 88: 669-673Google Scholar). With the exception of the K+ channel gene, this up-regulation appears to be induced primarily at the level of transcription (12Friday B.B. Pavlath G.K. J. Cell Sci. 2001; 114: 303-310Google Scholar, 13Shin K.S. Park J.-Y. Kwon H. Chung C.H. Kang M.-S. J. Biol. Chem. 1997; 272: 21227-21232Google Scholar, 14Freyssenet D. Di Carlo M. Hood D.A. J. Biol. Chem. 1999; 274: 9305-9311Google Scholar, 15Halseth A.E. O'Doherty R.M. Printz R.L. Bracy D.P. Granner D.K. Wasserman D.H. J. Appl. Physiol. 2000; 88: 669-673Google Scholar). In addition, recent evidence has implicated calcium signaling pathways in slow versus fast fiber gene expression. These studies have demonstrated that prolonged low amplitude increases in intracellular calcium, such as those produced by slow motoneurons, result in maximal activation of calcineurin, which in turn dephosphorylates members of the NFAT family of transcription factors, allowing them to activate the promoters of such slow-specific muscle genes as myoglobin and slow troponin I (16Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Google Scholar). Transgenic mouse studies have also implicated calcineurin as well as CaM kinase and the MEF-2 family of transcription factors in the specification of slow/oxidative gene expression (17Wu H. Naya F.J. McKinsey T.A. Mercer B. Shelton J.M. Chin E.R. Simard A.R. Michel R.N. Bassel-Duby R. Olson E.N. Williams R.S. EMBO J. 2000; 19: 1963-1973Google Scholar, 18Wu H. Kanatous S.B. Thurmond F.A. Gallardo T. Isotani E. Bassel-Duby R. Williams R.S. Science. 2002; 296: 349-352Google Scholar, 19Naya F.J. Mercer B. Shelton J. Richardson J.A. Williams R.S. Olson E.N. J. Biol. Chem. 2000; 275: 4545-4548Google Scholar), although studies using plasmid DNA injection failed to find a role for calcineurin in the specification of slow or fast muscle gene expression in vivo(20Swoap S.J. Hunter R.B. Stevenson E.J. Mitchell-Felton H. Kansagra N.V. Lang J.M. Esser K.A. Kandarian S.C. Am. J. Physiol. 2000; 279: C915-C924Google Scholar). Calcium-induced activation of calcineurin is also thought to underlie the fast-to-slow transitions in myosin heavy chain (MyHC) isoform expression, both in vitro and in vivo (21Kubis H.-P. Haller E.-A. Wetzel P. Gros G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4205-4210Google Scholar, 22Meisner Gros G. M. H.-P. J. Physiol. 2001; Scholar, U. J. P. Mol. Cell. Biol. 2000; Scholar, Mol. Biol. Cell. 2001; 12: Scholar, X. H. J. P. R. J. Biol. Chem. 2000; 275: Scholar, M. G. E. P. T. S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). is is calcium signaling is involved in the between fast fiber MyHC isoform transitions occur in a pattern during muscle adaptation, to IId/x to IIa to type I (1Pette D. Staron R.S. Int. Rev. Cytol. 1997; 170: 143-223Google Scholar). calcineurin involved in the fast to slow IIa to type I MyHC or is calcineurin signaling required to to IId/x and/or IId/x to The IIa MyHC isoform is in fibers with a oxidative more to that of type I fibers or IId/x to that IIa MyHC expression with type I We demonstrated that the promoter of the IIa MyHC gene activated to a by a active calcineurin the IId/x or MyHC that calcineurin signaling play a role in transitions between fast and that IIa MyHC with type I MyHC expression J. Biol. Chem. 2001; Scholar). is expression of the fast MyHC genes is also sensitive to intracellular calcium or signaling pathways are involved in transducing shifts in fast MyHC expression. We the of of the adult mouse skeletal MyHC promoters to calcium signaling in order to the role of intracellular calcium in shifts in fast fiber-specific gene expression. We that the adult skeletal MyHC promoter are sensitive to increased intracellular calcium, with IIa ≫ IId/x > IIb. We used overexpression and inhibitors to specific signaling pathways involved in calcium of the IIa MyHC promoter. that changes in intracellular calcium play a role in the shifts in fast MyHC expression during increased muscle activation and several intracellular pathways that be involved in this of of the mouse IId/x, and MyHC promoter J. Biol. Chem. 2001; Scholar). of the promoter for the IId/x, and MyHC genes the plasmid of the and NFAT in the IIa MyHC promoter using a of the MEF-2 and NFAT binding sites by whereas the for NFAT, SRF, and transcription also by whereas the by by the for active the of the IId/x MyHC promoter. on in and serum at using to the In the to for to of serum of myotubes with or of calcium for used cells in and of the cell for both promoter and activity using a are as by levels. myotubes with or for and and in at determined using the and to protein by using to using a and for at in and serum with a to the of CaM kinase II for at several in and for with an using a using and using R.M. Scholar). to and to for The and cells in and and and at for The cells in of and in of A. using a and at for The at for and in C and and with with a type B. The on for and at for The for The protein determined using the The nuclear at the or the NFAT of the IIa promoter with and used for of to of nuclear in a of and binding for at The on a at The and on a We the of the three adult fast MyHC promoters to increased intracellular calcium in for induced activation of both and mouse IIa MyHC promoters and the mouse IId/x and MyHC promoters the activity of the IIa MyHC promoter, which is associated with more oxidative fast to a by intracellular calcium the IId/x and MyHC We to IIa MyHC promoter activity be increased in response to of stimulation with the used in We used and of stimulation and IIa MyHC promoter activity at several stimulation of myotubes for these in a and increase in IIa MyHC promoter activity With of IIa promoter activity not activated at or but increased at and to the IIa MyHC promoter activation and by IIa MyHC promoter activity to With of IIa MyHC promoter activity increased of stimulation and increased a of stimulation at IIa MyHC promoter activity but at stimulation for produced a more and response with of stimulation and not to intracellular calcium at three signaling the calcineurin, CaM kinase, and protein kinase C We used inhibitors for each of these pathways to their role in ionophore-induced activation of the IIa MyHC promoter. In addition, we the effects of calcineurin on MyHC IIa expression. with an of calcineurin, activation of the IIa MyHC promoter in myotubes whereas overexpression of a activated of calcineurin increased IIa MyHC protein expression of myotubes with an of CaM kinase also ionophore-induced IIa MyHC promoter activity activation of CaM kinase II by calcium and the allowing to active of calcium levels. also in a increase in the of CaM kinase II at of but at In inhibitors of protein kinase C activity no significant effect on ionophore-induced activation of the IIa MyHC promoter to ionophore-induced IIa MyHC promoter activation, but the effect and not the effect at the of which is specific at We the effect of a more specific protein kinase C on ionophore-induced IIa MyHC promoter with no effect on ionophore-induced IIa MyHC promoter activity that protein kinase C is not involved in IIa MyHC promoter activity in response to increased intracellular these a role for calcineurin and CaM kinase II but not protein kinase C in calcium-induced activation of the IIa MyHC kinase and of IIa MyHC promoter myotubes with the IIa MyHC promoter with different of an of CaM kinase are as a of the response to with of the IIa promoter in a different for the of CaM kinase The a of of each kinase C and IIa MyHC promoter calcium with the IIa MyHC promoter and with different of an of protein kinase C. are as a of the response to with to IIa activation in response to calcium but the and not different effect of with a more specific of protein kinase C, at no effect on of the IIa MyHC We also the role of the MAP kinase pathways in ionophore-induced IIa MyHC promoter of myotubes with or inhibitors of the kinase and stimulation of IIa MyHC promoter whereas an of and a phosphatidylinositol not We also the effects of overexpression of MEKK1, an of the kinase on ionophore-induced IIa MyHC activation using both a and a promoter to expression. with ionophore-induced IIa MyHC promoter activation, whereas with ionophore-induced IIa MyHC promoter activity of with the also in a of myotubes whereas no effect on the effect of on ionophore-induced promoter activity to be ionophore-induced IIa MyHC promoter activity by whereas not and IIa promoter most through We the role of three transcription factor implicated in calcium signaling pathways NFAT, and on IIa MyHC promoter activation by binding sites for three factors are present in the IIa promoter J. Biol. Chem. 2001; Scholar). of or in a but of IIa MyHC promoter activity and as J. Biol. Chem. 2001; Scholar), whereas with in a increase in IIa MyHC promoter activity with of or in a significant with and In with not ionophore-induced IIa MyHC promoter activity with with of MEF-2 transcription factors by CaM kinase is thought to be through and of II the T.A. J. Olson E.N. 2000; Scholar). We the role of on activation of the IIa MyHC promoter. with or in a significant of ionophore-induced IIa MyHC activation with the MEF-2 these a role for MEF-2 as well as NFAT in ionophore-induced IIa MyHC promoter We the effect of MEF-2 and/or NFAT a one to binding of these transcription factors to the IIa MyHC promoter or an of MEF-2 and/or NFAT activation of this we used binding sites for MEF-2, NFAT, and with the activity of the MEF-2 and NFAT increased by but activity not these a role for MEF-2 and NFAT but not in ionophore-induced activation of the IIa MyHC promoter. We used to the binding sites for these factors the IIa MyHC promoter and with these to their role in ionophore-induced or of the or of the NFAT in a significant of ionophore-induced IIa MyHC promoter activation of the MEF-2 and of the NFAT in the not ionophore-induced IIa MyHC promoter activity that sites to this of the no effect on ionophore-induced activation of the IIa MyHC promoter We in changes in the binding activity of MEF-2 and/or NFAT to their binding sites on the promoter. using the of the IIa MyHC promoter demonstrated increased of three with nuclear myotubes with myotubes on J. Biol. Chem. 1998; Scholar), and A. the binding activity is a to binding of MEF-2 family members in the whereas the The of a MEF-2 in a of the in the The a protein identified by J. Biol. Chem. 1998; Scholar). of three protein is in nuclear with nuclear using the NFAT binding binding activity the MEF-2 is at a that NFAT binding is MEF-2 binding in both and on studies on NFAT is that the binding activity is NFAT, whereas the binding activity J. Biol. Chem. 2001; Scholar). The binding activity of NFAT is increased in nuclear with nuclear but the is not as as that for MEF-2 we used an a binding for the transcription factor to the this In to the using the or NFAT binding of not increased by in for the One of the most prominent to be involved in fiber-specific gene expression is intracellular calcium levels. In the present work, we determined expression of the adult fast skeletal MyHC genes is sensitive to increases in intracellular the three adult fast MyHC IIa the by to by IId/x and The increase in IId/x MyHC promoter activity is in to by Gros G. M. H.-P. J. Physiol. 2001; Scholar), demonstrated a in IId/x MyHC and protein in response to prolonged of myotubes in In this myotubes with for of whereas in the present we used of at of the between the of Gros G. M. H.-P. J. Physiol. 2001; Scholar) and the present be the in the IId/x MyHC gene be activated during the of to increased intracellular calcium levels but be as myotubes a more oxidative studies have demonstrated that overexpression of calcineurin in myotubes increases slow MyHC protein levels and not or fast MyHC expression U. J. P. Mol. Cell. Biol. 2000; Scholar, Mol. Biol. Cell. 2001; 12: Scholar). the three different fast MyHC not in those be determined shifts the fast fiber and/or IId/x MyHC have type IIa MyHC in these we demonstrated that active calcineurin activated the IIa MyHC promoter, by with for the IId/x and promoters J. Biol. Chem. 2001; Scholar). we demonstrate that calcineurin overexpression also increases expression of IIa MyHC protein the IIa MyHC promoter is activated to a the fast MyHC promoters by intracellular calcium levels in a These that signaling be involved in the IId/x and MyHC of MyHC associated with more oxidative fibers I and that are to the involved in prolonged muscle using have that the and CaM signaling pathways are involved in fast-to-slow fiber type transitions (16Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Google Scholar, H. Naya F.J. McKinsey T.A. Mercer B. Shelton J.M. Chin E.R. Simard A.R. Michel R.N. Bassel-Duby R. Olson E.N. Williams R.S. EMBO J. 2000; 19: 1963-1973Google Scholar, 18Wu H. Kanatous S.B. Thurmond F.A. Gallardo T. Isotani E. Bassel-Duby R. Williams R.S. Science. 2002; 296: 349-352Google Scholar, 19Naya F.J. Mercer B. Shelton J. Richardson J.A. Williams R.S. Olson E.N. J. Biol. Chem. 2000; 275: 4545-4548Google Scholar). these studies these factors activated type I expression or involved in transitions between fast vivo using calcineurin inhibitors has that of calcineurin a type I to type IIa MyHC expression X. H. J. P. R. J. Biol. Chem. 2000; 275: Scholar), but this in the muscle, which is type and not the of fast fiber types. The present that intracellular calcium and calcineurin are involved in IIa MyHC expression and be involved in the or IId/x fibers to with this signaling most active in the oxidative muscle fibers in vivo IId/x and during to increased Simard A.R. Bassel-Duby R. Williams R.S. Michel R.N. J. Biol. Chem. 2001; Scholar), whereas of with the calcineurin MyHC expression type I to type IId/x and M. G. E. P. T. S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). We the role of pathways activated by increased intracellular calcium levels on MyHC promoter CaM kinase and protein kinase C. that CaM kinase II an role in IIa MyHC promoter activation In different protein kinase C a significant effect on IIa MyHC promoter by kinase C has been to slow MyHC expression in myotubes in a but not to fast MyHC expression Dev. 1999; Scholar), with the present We also that and play a role in IIa MyHC promoter activation in response to The MAP kinase signaling pathways play critical roles in many cell but are prominent in cell of the cells in myotubes to with or is that the to effects of the inhibitors on cell These that the kinase calcium activation of the IIa MyHC gene muscle the kinase are active in both and in with a role for this in muscle gene expression K.S. F. M. Mol. Cell. Biol. 2000; Scholar). In addition, and which of and induce slow MyHC expression in muscle in vivo M. E. G. T. S. Cell Biol. 2000; Scholar). is an of the kinase Inhibition of by in the in calcium of the IIa promoter, which in turn that molecules specific to are for of the IIa MyHC the effects of its effects on cell using the IId/x MyHC promoter to expression, ionophore-induced IIa that the kinase be involved in IIa MyHC activation in In p38 phosphatidylinositol appears to play a role in the activation of the IIa MyHC promoter. The of a role for p38 that p38 activity of MEF-2 family members M. H. F. Olson E.N. J. Mol. Cell. Biol. 1999; 19: Scholar). are also at with those of U. J. P. Mol. Cell. Biol. 2000; Scholar), that of myotubes with a an of in up-regulation of fast MyHC protein expression. of p38 are in muscle, and has been that not p38 M. H. F. Olson E.N. J. Mol. Cell. Biol. 1999; 19: Scholar), which the effects in the present We present several of that the MEF-2 and NFAT of transcription factors are involved in the IIa MyHC promoter in response to with the on the role of the on slow fiber gene expression (16Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Google Scholar, H. Naya F.J. McKinsey T.A. Mercer B. Shelton J.M. Chin E.R. Simard A.R. Michel R.N. Bassel-Duby R. Olson E.N. Williams R.S. EMBO J. 2000; 19: 1963-1973Google Scholar, 18Wu H. Kanatous S.B. Thurmond F.A. Gallardo T. Isotani E. Bassel-Duby R. Williams R.S. Science. 2002; 296: 349-352Google Scholar), these are with the that MEF-2 and NFAT transcription factors are involved in the activation of genes associated with a more oxidative such as type I and type IIa In we found no evidence to that is associated with ionophore-induced IIa MyHC promoter The transcription factor has been implicated in calcium-induced shifts in and gene transcription (4Miranti C.K. Ginty D.D. Huang G. Chatila T. Greenberg M.E. Mol. Cell. Biol. 1995; 15: 3672-3684Google Scholar, 6McDonough P.M. Hanford D.S. Sprenkle A.B. Mellon N.R. Glembotski C.C. J. Biol. Chem. 1997; 272: 24046-24053Google Scholar, 7Bernal-Mizrachi E. Wice B. Inoue H. Permutt M.A. J. Biol. Chem. 2000; 275: 25681-25689Google Scholar). is also in a in skeletal muscle M. J.A. J. Appl. Physiol. 1999; Scholar, M. J. Appl. Physiol. 2001; Scholar) and several genes, including P. N. J. Biol. Chem. 2001; Scholar) and W. Am. J. Physiol. 2000; Scholar). In addition, is by CaM kinase II at several sites M. Biochem. 2000; Scholar). not to be critical for IIa MyHC promoter activation in response to has been that changes in the expression of muscle genes result the effect of exercise on signaling pathways to increases in gene these are at as during a training these increases in levels a which changes in protein levels occur that changes the muscle Am. J. Physiol. Scholar). this has been demonstrated most for metabolic genes such as the and various genes Am. J. Physiol. H. B. Am. J. Physiol. 2000; 279: Scholar). exercise has been to activate signaling in vivo H. B. Kanatous S. P. Naya F.J. Shelton J.M. J.A. J.M. Olson E.N. Bassel-Duby R. Williams R.S. EMBO J. 2001; Scholar), whereas in also induces the kinase and kinase signaling pathways S. T. J. Physiol. 2000; Scholar, M. E. A. J. Physiol. 2001; Scholar). In the studies we myotubes with calcium for in order to the maximal response to increased intracellular to of increased intracellular calcium that more of exercise also IIa MyHC promoter we myotubes for or and found that this in a increase in IIa MyHC promoter activity that and with for in a increase in IIa MyHC promoter activity that to by for in a increase in IIa MyHC promoter and activity These that stimulation also result in up-regulation of IIa MyHC expression and are with the that changes in gene expression are a of the of skeletal muscle to increased We Olson for the of various expression for with the calcineurin and for in the